Inbred corn line RBO1

ABSTRACT

An inbred corn line, designated RBO1, is disclosed. The invention relates to the seeds of inbred corn line RBO1, to the plants of inbred corn line RBO1 and to methods for producing a corn plant, either inbred or hybrid, by crossing the inbred line RBO1 with itself or another corn line. The invention further relates to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other inbred corn lines derived from the inbred RBO1.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a new and distinctive corninbred line, designated RBO1. There are numerous steps in thedevelopment of any novel, desirable plant germplasm. Plant breedingbegins with the analysis and definition of problems and weaknesses ofthe current germplasm, the establishment of program goals, and thedefinition of specific breeding objectives. The next step is selectionof germplasm that possess the traits to meet the program goals. The goalis to combine in a single variety or hybrid an improved combination ofdesirable traits from the parental germplasm. These important traits mayinclude higher yield, resistance to diseases and insects, better stalksand roots, tolerance to drought and heat, and better agronomic quality.With mechanical harvesting of many crops, uniformity of plantcharacteristics such as germination and stand establishment, growthrate, maturity and plant and ear height is important.

[0002] Choice of breeding or selection methods depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc.). For highly heritable traits, a choice ofsuperior individual plants evaluated at a single location will beeffective, whereas for traits with low heritability, selection should bebased on mean values obtained from replicated evaluations of families ofrelated plants. Popular selection methods commonly include pedigreeselection, modified pedigree selection, mass selection, and recurrentselection.

[0003] The complexity of inheritance influences choice of the breedingmethod. Backcross breeding is used to transfer one or a few favorablegenes for a highly heritable trait into a desirable cultivar. Thisapproach has been used extensively for breeding disease-resistantcultivars. Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

[0004] Each breeding program should include a periodic, objectiveevaluation of the efficiency of the breeding procedure. Evaluationcriteria vary depending on the goal and objectives, but should includegain from selection per year based on comparisons to an appropriatestandard, overall value of the advanced breeding lines, and number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

[0005] Promising advanced breeding lines are thoroughly tested andcompared to appropriate standards in environments representative of thecommercial target area(s) for three years at least. The best lines arecandidates for new commercial cultivars; those still deficient in a fewtraits are used as parents to produce new populations for furtherselection.

[0006] These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

[0007] A most difficult task is the identification of individuals thatare genetically superior, because for most traits the true genotypicvalue is masked by other confounding plant traits or environmentalfactors. One method of identifying a superior plant is to observe itsperformance relative to other experimental plants and to a widely grownstandard cultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

[0008] The goal of plant breeding is to develop new, unique and superiorcorn inbred lines and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations. The breeder has no direct control at the cellularlevel. Therefore, two breeders will never develop the same line, or evenvery similar lines, having the same corn traits.

[0009] Each year, the plant breeder selects the germplasm to advance tothe next generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred lineswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop a superior new corn inbred line.

[0010] The development of commercial corn hybrids requires thedevelopment of homozygous inbred lines, the crossing of these lines, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop inbred lines from breedingpopulations. Breeding programs combine desirable traits from two or moreinbred lines or various broad-based sources into breeding pools fromwhich inbred lines are developed by selfing and selection of desiredphenotypes. The new inbreds are crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which havecommercial potential.

[0011] Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

[0012] Mass and recurrent selections can be used to improve populationsof either self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

[0013] Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

[0014] Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., “Principles of Plant Breeding” John Wiley and Son, pp.115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

[0015] Proper testing should detect any major faults and establish thelevel of superiority or improvement over current cultivars. In additionto showing superior performance, there must be a demand for a newcultivar that is compatible with industry standards or which creates anew market. The introduction of a new cultivar will incur additionalcosts to the seed producer, the grower, processor and consumer; forspecial advertising and marketing, altered seed and commercialproduction practices, and new product utilization. The testing precedingrelease of a new cultivar should take into consideration research anddevelopment costs as well as technical superiority of the finalcultivar. For seed-propagated cultivars, it must be feasible to produceseed easily and economically.

[0016] Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F₁progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F₁ hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor exhibited by F₁ hybrids islost in the next generation (F₂). Consequently, seed from hybridvarieties is not used for planting stock.

[0017] Hybrid corn seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twocorn inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

[0018] The laborious, and occasionally unreliable, detasseling processcan be avoided by using cytoplasmic male-sterile (CMS) inbreds. Plantsof a CMS inbred are male sterile as a result of factors resulting fromthe cytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent incorn plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile corn and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

[0019] There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Theseand all patents referred to are incorporated by reference. In additionto these methods, Albertsen et al., U.S. Pat. No. 5,432,068 havedeveloped a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility, silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

[0020] There are many other methods of conferring genetic male sterilityin the art, each with its own benefits and drawbacks. These methods usea variety of approaches such as delivering into the plant a geneencoding a cytotoxic substance associated with a male tissue specificpromoter or an anti-sense system in which a gene critical to fertilityis identified and an antisense to that gene is inserted in the plant(see, Fabinjanski, et al. EPO 89/3010153.8 publication no. 329, 308 andPCT application PCT/CA90/00037 published as WO 90/08828).

[0021] Another version useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, G. R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application and genotype specificallyoften limit the usefulness of the approach.

[0022] Corn is an important and valuable field crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding corn hybridsthat are agronomically sound. The reasons for this goal are obviously tomaximize the amount of grain produced on the land used and to supplyfood for both animals and humans. To accomplish this goal, the cornbreeder must select and develop corn plants that have the traits thatresult in superior parental lines for producing hybrids.

SUMMARY OF THE INVENTION

[0023] According to the invention, there is provided a novel inbred cornline, designated RBO1. This invention thus relates to the seeds ofinbred corn line RBO1, to the plants of inbred corn line RBO1 and tomethods for producing a corn plant produced by crossing the inbred lineRBO1 with itself or another corn line, and to methods for producing acorn plant containing in its genetic material one or more transgenes andto the transgenic corn plants produced by that method. This inventionalso relates to methods for producing other inbred corn lines derivedfrom inbred corn line RBO1 and to the inbred corn lines derived by theuse of those methods. This invention further relates to hybrid cornseeds and plants produced by crossing the inbred line RBO1 with anothercorn line.

[0024] The inbred corn plant of the invention may further comprise, orhave, a cytoplasmic factor or other factor that is capable of conferringmale sterility. Parts of the corn plant of the present invention arealso provided, such as e.g., pollen obtained from an inbred plant and anovule of the inbred plant.

[0025] In another aspect, the present invention provides regenerablecells for use in tissue culture or inbred corn plant RBO1. The tissueculture will preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing inbredcorn plant, and of regenerating plants having substantially the samegenotype as the foregoing inbred corn plant. Preferably, the regenerablecells in such tissue cultures will be embryos, protoplasts, meristematiccells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks or stalks. Still further, the presentinvention provides corn plants regenerated from the tissue cultures ofthe invention.

[0026] Another objective of the invention is to provide methods forproducing other inbred corn plants derived from inbred corn line RBO1.Inbred corn lines derived by the use of those methods are also part ofthe invention.

[0027] The invention also relates to methods for producing a corn plantcontaining in its genetic material one or more transgenes and to thetransgenic corn plant produced by that method.

[0028] In another aspect, the present invention provides for single geneconverted plants of RBO1. The single transferred gene may preferably bea dominant or recessive allele. Preferably, the single transferred genewill confer such trait as male sterility, herbicide resistance, insectresistance, resistance for bacterial, fungal, or viral disease, malefertility, and enhanced nutritional quality. The single gene may be anaturally occurring maize gene or a transgene introduced through geneticengineering techniques.

[0029] The invention further provides methods for developing corn plantin a corn plant breeding program using plant breeding techniqueincluding recurrent selection, backcrossing, pedigree breeding,restriction fragment length polymorphism enhanced selection, geneticmarker enhanced selection and transformation. Seeds, corn plant, andparties thereof produced by such breeding methods are also part of theinvention.

DEFINITIONS

[0030] In the description and tables which follow, a number of terms areused. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

[0031] Allele. The allele is any of one or more alternative form of agene, all of which alleles relates to one trait or characteristic. In adiploid cell or organism, the two alleles of a given gene occupycorresponding loci on a pair of homologous chromosomes.

[0032] Backcrossing. Backcrossing is a process in which a breederrepeatedly crosses hybrid progeny back to one of the parents, forexample, a first generation hybrid F₁ with one of the parental genotypeof the F₁ hybrid.

[0033] Essentially all the physiological and morphologicalcharacteristics. A plant having essentially all the physiological andmorphological characteristics means a plant having the physiological andmorphological characteristics, except for the characteristics derivedfrom the converted gene.

[0034] Regeneration. Regeneration refers to the development of a plantfrom tissue culture.

[0035] Single gene converted. Single gene converted or conversion plantrefers to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single gene transferred into the inbred via the backcrossingtechnique or via genetic engineering.

[0036] Predicted RM. This trait for a hybrid, predicted relativematurity (RM), is based on the harvest moisture of the grain. Therelative maturity rating is based on a known set of checks and utilizesconventional maturity such as the Comparative Relative Maturity RatingSystem or its similar, the Minnesota Relative Maturity Rating System.

[0037] MN RM. This represents the Minnesota Relative Maturity Rating (MNRM) for the hybrid and is based on the harvest moisture of the grainrelative to a standard set of checks of previously determined MN RMrating. Regression analysis is used to compute this rating.

[0038] Yield (Quintals/Hectare). The yield is the actual yield of thegrain at harvest adjusted to 15.5% moisture.

[0039] Moisture. The moisture is the actual percentage moisture of thegrain at harvest.

[0040] GDU Silk. The GDU silk (=heat unit silk) is the number of growingdegree units (GDU) or heat units required for an inbred line or hybridto reach silk emergence from the time of planting. Growing degree unitsare calculated by the Barger Method, where the heat units for a 24-hourperiod are: GDU=((Max Temp+Min Temp)/2)−50 The highest maximum used is86

F and the lowest minimum used is 50

F. For each hybrid, it takes a certain number of GDUs to reach variousstages of plant development. GDUs are a way of measuring plant maturity.

[0041] Stalk Lodging. This is the percentage of plants that stalk lodge,i.e., stalk breakage, as measured by either natural lodging or pushingthe stalks determining the percentage of plants that break off below theear. This is a relative rating of a hybrid to other hybrids forstandability.

[0042] Root Lodging. The root lodging is the percentage of plants thatroot lodge; i.e., those that lean from the vertical axis at anapproximate 30

angle or greater would be counted as root lodged.

[0043] Plant Height. This is a measure of the height of the hybrid fromthe ground to the tip of the tassel, and is measured in centimeters.

[0044] Ear Height. The ear height is a measure from the ground to theear node attachment, and is measured in centimeters.

[0045] Dropped Ears. This is a measure of the number of dropped ears perplot, and represents the percentage of plants that dropped an ear priorto harvest.

[0046] Stay Green. Stay green is the measure of plant health near thetime of black layer formation (physiological maturity). A high scoreindicates better late-season plant health.

DETAILED DESCRIPTION OF THE INVENTION

[0047] Inbred corn line RBO1 is a yellow dent corn with superiorcharacteristics, and provides an excellent parental line in crosses forproducing first generation (F₁) hybrid corn. Inbred corn line RBO1 isbest adapted to the areas of the United States corn belt with a latitudeof greater than 38

north and can be used to produce hybrids having a relative maturity ofapproximately 95-113 on the Comparative Relative Maturity Rating Systemfor harvest moisture of grain. Inbred corn line RBO1 shows an excellentseedling vigor, an early pollen shed, excellent brittle stalkresistance, superior root and stalk strength, and above average staygreen.

[0048] RBO1 is similar to SBB1, however there are numerous differencesincluding the fact that RBO1 flowers slightly earlier than SBB1. Hybridswith RBO1 also are lower eared and have a higher test weight.

[0049] RBO1 has a plant height of 202 cm with an average ear insertionof 57 cm. The kernels are arranged in distinct, slightly curved rows onthe ear. Heat units to 50% pollen shed are approximately 1412 and to 50%silk are approximately 1427.

[0050] RBO1 is an inbred line with very high yield potential and verystrong stalk and roots in hybrids. For an inbred of its maturity, RBO1results in a lower ear position in hybrid combination. Often thesehybrid combinations results in plants which are of much better thanaverage overall health when compared to inbred lines of similarmaturity.

[0051] Some of the criteria used to select ears in various generationsinclude: yield, stalk quality, root quality, disease tolerance, lateplant greenness, late season plant intactness, ear retention, earheight, pollen shedding ability, silking ability, and corn borertolerance. During the development of the line, crosses were made toinbred testers for the purpose of estimating the line's general andspecific combining ability, and parallel evaluations were run in the USAby the Kirkland, Ill. Research Station. The inbred was evaluated furtheras a line and in numerous crosses by the Kirkland station and otherresearch stations across the Corn Belt. The inbred has proven to have agood combining ability in hybrid combinations.

[0052] The inbred line has shown uniformity and stability for thetraits, within the limits of environmental influence for the traits. Ithas been self-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in RBO1.

[0053] Inbred corn line RBO1 has the following morphologic and othercharacteristics (based primarily on data collected at Kirkland, Ill.).

VARIETY DESCRIPTION INFORMATION

[0054] TYPE: Dent

[0055] REGION WHERE DEVELOPED: Northcentral Illinois.

[0056] MATURITY: 101 Days Days Heat Units From emergence to 50% ofplants in silk: 83 1427 From emergence to 50% of plants in pollen: 821412

[0057] PLANT:

[0058] Plant Height to tassel tip: 201.7 cm (Standard Deviation=4.90)

[0059] Ear Height to base of top ear 56.9 cm (5.45)

[0060] Average Length of Top Ear Internode: 11.2 cm (0.26)

[0061] Average number of Tillers: 0.0 (0.00)

[0062] Average Number of Ears per Stalk: 1.0 (0.00)

[0063] Anthocyanin of Brace Roots: Absent

[0064] LEAF:

[0065] Width of Ear Node Leaf: 7.9 cm (0.16)

[0066] Length of Ear Node Leaf: 82.3 cm (0.96)

[0067] Number of leaves above top ear: 5.9 (0.57)

[0068] Leaf Angle (from 2nd Leaf above ear at anthesis to Stalk aboveleaf): 75.8

(2.44)

[0069] Leaf Color: Dark Green Munsell Code 7.5 GY 4/4

[0070] Leaf Sheath Pubescence (Rate on scale from 1=none to 9=like peachfuz): 8

[0071] Marginal Waves (Rate on scale from 1=none to 9=many): 8

[0072] Longitudinal Creases (Rate on scale from 1=none to 9=many): 7

[0073] TASSEL:

[0074] Number of Lateral Branches: 7.6 (2.01)

[0075] Branch Angle from Central Spike: 41.9 (1.85)

[0076] Tassel Length (from top leaf collar to tassel top): 46.1 cm(1.20)

[0077] Pollen Shed (Rate on scale from 0=male sterile to 9=heavy shed):6

[0078] Anther Color: Light Purple to Pink Munsell Code 5 YR 4/2

[0079] Glume Color: Light Green Munsell Code 5GY 6/6

[0080] Bar Glumes: Absent

[0081] EAR: (Unhusked Data)

[0082] Silk Color (3 days after emergence): Greenish-Yellow Munsell Code2.5 GY 8/8

[0083] Fresh Husk Color (25 days after 50% silking): Medium GreenMunsell Code 5 GY 5/4

[0084] Dry Husk Color (65 days after 50% silking): Dark ta n MunsellCode 2.5 GY 7/4

[0085] Position of Ear: Upright

[0086] Husk Tightness (Rate on scale from 1=very loose to 9=very tight):4

[0087] Husk Extension at harvest: Short

[0088] EAR: (Husked Ear Data)

[0089] Ear Length: 13.5 cm (0.97)

[0090] Ear Diameter at mid-point: 41.4 mm (1.15)

[0091] Ear Weight: 111.6 gm (10.01)

[0092] Number of Kernel Rows: 15.4 (0.97)

[0093] Kernel Rows: Distinct

[0094] Row Alignment: Slightly Curved

[0095] Shank Length: 14.1 cm (3.73)

[0096] Ear Taper: Average

[0097] KERNEL: (Dried)

[0098] Kernel Length: 11.2 mm (0.51)

[0099] Kernel Width: 7.2 mm (0.43)

[0100] Kernel Thickness: 4.4 mm (0.51)

[0101] Round Kernels (Shape Grade): 43%

[0102] Aleurone Color Pattern: Homozygous

[0103] Aleurone Color: Colorless

[0104] Hard Endosperm Color: Yellow Munsell code 2.5Y 8/8

[0105] Endosperm Type: Normal Starch

[0106] Weight per 100 kernels (unsized sample): 25.1 gm (1.45)

[0107] COB:

[0108] Cob Diameter at Mid-Point: 23.7 mm (0.72)

[0109] Cob Color: Red Munsell code 10 R 4/10

[0110] AGRONOMIC TRAITS:

[0111] Stay Green (at 65 days after anthesis) (Rate on scale from1=worst to 9=excellent): 7

[0112] 0% Dropped Ears (at 65 days after anthesis)

[0113] 0% Pre-anthesis Brittle Snapping

[0114] 0% Pre-anthesis Root Lodging

[0115] 0% Post-anthesis Root Lodging (at 65 days after anthesis)

[0116] Yield of Inbred Per Se (at 12-13% grain moisture): 59 Bu/ Acre

FURTHER EMBODIMENTS OF THE INVENTION

[0117] This invention also is directed to methods for producing a cornplant by crossing a first parent corn plant with a second parent cornplant wherein either the first or second parent corn plant is an inbredcorn plant of the line RBO1. Further, both first and second parent cornplants can come from the inbred corn line RBO1. Still further, thisinvention also is directed to methods for producing an inbred corn lineRBO1-derived corn plant by crossing inbred corn line RBO1 with a secondcorn plant and growing the progeny seed, and repeating the crossing andgrowing steps with the inbred corn line RBO1-derived plant from 0 to 7times. Thus, any such methods using the inbred corn line RBO1 are partof this invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using inbred corn lineRBO1 as a parent are within the scope of this invention, includingplants derived from inbred corn line RBO1. Advantageously, the inbredcorn line is used in crosses with other, different, corn inbreds toproduce first generation (F₁) corn hybrid seeds and plants with superiorcharacteristics.

[0118] It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

[0119] As used herein, the term plant includes plant cells, plantprotoplasts, plant cell tissue cultures from which corn plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,kernels, ears, cobs, leaves, husks, stalks, roots, root tips, anthers,silk and the like.

[0120] Duncan, et al., Planta 165:322-332 (1985) reflects that 97% ofthe plants cultured that produced callus were capable of plantregeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus that produced plants. In a further studyin 1988, Songstad, et al., Plant Cell Reports 7:262-265 (1988), reportsseveral media additions that enhance regenerability of callus of twoinbred lines. Other published reports also indicated that“nontraditional” tissues are capable of producing somatic embryogenesisand plant regeneration. K. P. Rao et al., Maize Genetics CooperationNewsletter, 60:64-65 (1986), refers to somatic embryogenesis from glumecallus cultures and B. V. Conger, et al., Plant Cell Reports, 6:345-347(1987) indicates somatic embryogenesis from the tissue cultures of cornleaf segments. Thus, it is clear from the literature that the state ofthe art is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

[0121] Tissue culture of corn is described in European PatentApplication, publication 160,390, incorporated herein by reference. Corntissue culture procedures are also described in Green and Rhodes, “PlantRegeneration in Tissue Culture of Maize,” Maize for Biological Research(Plant Molecular Biology Association, Charlottesville, Va. 367-372,(1982)) and in Duncan et al., “The Production of Callus Capable of PlantRegeneration from Immature Embryos of Numerous Zea Mays Genotypes,” 165Planta 322:332 (1985). Thus, another aspect of this invention is toprovide cells which upon growth and differentiation produce corn plantshaving the physiological and morphological characteristics of inbredcorn line RBO1.

[0122] The utility of inbred corn line RBO1 also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Croix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Potentially suitable for crosses with RBO1 may be the variousvarieties of grain sorghum, Sorghum bicolor (L.) Moench.

[0123] With the advent of molecular biological techniques that haveallowed the isolation and characterization of genes that encode specificprotein products, scientists in the field of plant biology developed astrong interest in engineering the genome of plants to contain andexpress foreign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed inbred line.

[0124] Plant transformation involves the construction of an expressionvector which will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed corn plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the corn plant(s).

Expression Vectors for Corn Transformation

[0125] Marker Genes—Expression vectors include at least one geneticmarker, operably linked to a regulatory element (a promoter, forexample) that allows transformed cells containing the marker to beeither recovered by negative selection, i.e., inhibiting growth of cellsthat do not contain the selectable marker gene, or by positiveselection, i.e., screening for the product encoded by the geneticmarker. Many commonly used selectable marker genes for planttransformation are well known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent which may be an antibiotic or a herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. A few positive selection methods are also known in the art.

[0126] One commonly used selectable marker gene for plant transformationis the neomycin phosphotransferase II (nptII) gene, isolated fromtransposon Tn5, which when placed under the control of plant regulatorysignals confers resistance to kanamycin. Fraley et al., Proc. Natl.Acad. Sci. U.S.A., 80:4803 (1983). Another commonly used selectablemarker gene is the hygromycin phosphotransferase gene which confersresistance to the antibiotic hygromycin. Vanden Elzen et al., Plant Mol.Biol., 5:299 (1985).

[0127] Additional selectable marker genes of bacterial origin thatconfer resistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant PhysioL86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

[0128] Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

[0129] Another class of marker genes for plant transformation requirescreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS,β-galactosidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984). Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247:449 (1990).

[0130] Recently, in vivo methods for visualizing GUS activity that donot require destruction of plant tissue have been made available.Molecular Probes publication 2908, Imagene Green™, p. 1-4 (1993) andNaleway et al., J. Cell Biol. 115:151a (1991). However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

[0131] More recently, a gene encoding Green Fluorescent Protein (GFP)has been utilized as a marker for gene expression in prokaryotic andeukaryotic cells. Chalfie et al., Science 263:802 (1994). GFP andmutants of GFP may be used as screenable markers.

[0132] Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

[0133] As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

[0134] An inducible promoter is operably linked to a gene for expressionin corn. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn. With an inducible promoter the rate oftranscription increases in response to an inducing agent.

[0135] Any inducible promoter can be used in the instant invention. SeeWard et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

[0136] A constitutive promoter is operably linked to a gene forexpression in corn or the constitutive promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn.

[0137] Many different constitutive promoters can be utilized in theinstant invention. Exemplary constitutive promoters include, but are notlimited to, the promoters from plant viruses such as the 35S promoterfrom CaMV (Odell et al., Nature 313:810-812 (1985) and the promotersfrom such genes as rice actin (McElroy et al., Plant Cell 2:163-171(1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632(1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU(Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten etal., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al.,Mol. Gen. Genetics 231:276-285 (1992) and Atanassova et al., PlantJournal 2 (3): 291-300 (1992)).

[0138] The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napusALS3 structural gene (or a nucleotide sequence similarity to saidXba1/Ncol fragment), represents a particularly useful constitutivepromoter. See PCT application WO96/30530.

C. Tissue-specific or Tissue-preferred Promoters

[0139] A tissue-specific promoter is operably linked to a gene forexpression in corn. Optionally, the tissue-specific promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in corn. Plants transformedwith a gene of interest operably linked to a tissue-specific promoterproduce the protein product of the transgene exclusively, orpreferentially, in a specific tissue.

[0140] Any tissue-specific or tissue-preferred promoter can be utilizedin the instant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

[0141] Transport of protein produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall or mitochondroin or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine, during protein synthesis andprocessing, where the encoded protein is ultimately compartmentalized.

[0142] The presence of a signal sequence directs a polypeptide to eitheran intracellular organelle or subcellular compartment or for secretionto the apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

[0143] With transgenic plants according to the present invention, aforeign protein can be produced in commercial quantities. Thus,techniques for the selection and propagation of transformed plants,which are well understood in the art, yield a plurality of transgenicplants which are harvested in a conventional manner, and a foreignprotein then can be extracted from a tissue of interest or from totalbiomass. Protein extraction from plant biomass can be accomplished byknown methods which are discussed, for example, by Heney and Orr, Anal.Biochem. 114:92-6 (1981).

[0144] According to a preferred embodiment, the transgenic plantprovided for commercial production of foreign protein is corn. Inanother preferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

[0145] Likewise, by means of the present invention, agronomic genes canbe expressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

[0146] 1. Genes That Confer Resistance to Pests or Disease and ThatEncode:

[0147] A. Plant disease resistance genes. Plant defenses are oftenactivated by specific interaction between the product of a diseaseresistance gene (R) in the plant and the product of a correspondingavirulence (Avr) gene in the pathogen. A plant inbred line can betransformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.,Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistanceto Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. Tomato encodes aprotein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2gene for resistance to Pseudomonas syringae).

[0148] B. A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

[0149] C. A lectin. See, for example, the disclose by Van Damme et al.,Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

[0150] D. A vitamin-binding protein such as avidin. See PCT applicationU.S. Ser. No. 93/06487, the contents of which are hereby incorporated byreference. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

[0151] E. An enzyme inhibitor, for example, a protease or proteinaseinhibitor or an amylase inhibitor. See, for example, Abe et al., J.Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice cysteineproteinase inhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993)(nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I),Sumitani et al., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotidesequence of Streptomyces nitrosporeusα-amylase inhibitor).

[0152] F. An insect-specific hormone or pheromone such as an ecdysteroidand juvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

[0153] G. An insect-specific peptide or neuropeptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of Regan, J. Biol. Chem. 269:9 (1994) (expressioncloning yields DNA coding for insect diuretic hormone receptor), andPratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989) (anallostatin is identified in Diploptera puntata). See also U.S. Pat. No.5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

[0154] H. An insect-specific venom produced in nature by a snake, awasp, etc. For example, see Pang et al., Gene 116:165 (1992), fordisclosure of heterologous expression in plants of a gene coding for ascorpion insectotoxic peptide.

[0155] I. An enzyme responsible for a hyper accumulation of amonterpene, a sesquiterpene, a steroid, hydroxamic acid, aphenylpropanoid derivative or another non-protein molecule withinsecticidal activity.

[0156] J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

[0157] K. A molecule that stimulates signal transduction. For example,see the disclosure by Botella et al., Plant Molec. Biol. 24:757 (1994),of nucleotide sequences for mung bean calmodulin cDNA clones, and Griesset al., Plant Physiol. 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

[0158] L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

[0159] M. A membrane permease, a channel former or a channel blocker.For example, see the disclosure of Jaynes et al., Plant Sci 89:43(1993), of heterologous expression of a cecropin-β, lytic peptide analogto render transgenic tobacco plants resistant to Pseudomonassolanacearum.

[0160] N. A viral-invasive protein or a complex toxin derived therefrom.For example, the accumulation of viral coat proteins in transformedplant cells imparts resistance to viral infection and/or diseasedevelopment effected by the virus from which the coat protein gene isderived, as well as by related viruses. See Beachy et al., Ann. rev.Phytopathol. 28:451 (1990). Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

[0161] O. An insect-specific antibody or an immunotoxin derivedtherefrom. Thus, an antibody targeted to a critical metabolic functionin the insect gut would inactivate an affected enzyme, killing theinsect. Cf. Taylor et al., Abstract#497, Seventh Int'l Symposium onMolecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

[0162] P. A virus-specific antibody. See, for example, Tavladoraki etal., Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

[0163] Q. A developmental-arrestive protein produced in nature by apathogen or a parasite. Thus, fungal endo α-1, 4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-α-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10:1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2:367 (1992).

[0164] R. A development-arrestive protein produced in nature by a plant.For example, Logemann et al., Bioi/Technology 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

[0165] 2. Genes That Confer Resistance to a Herbicide, For Example:

[0166] A. A herbicide that inhibits the growing point or meristem, suchas an imidazalinone or a sulfonylurea. Exemplary genes in this categorycode for mutant ALS and AHAS enzyme as described, for example, by Lee etal., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

[0167] B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

[0168] C. A herbicide that inhibits photosynthesis, such as a triazine(psbA and gs+genes) and a benzonitrile (nitrilase gene). Przibilla etal., Plant Cell 3:169 (1991), describe the transformation ofChlamydomonas with plasmids encoding mutant psbA genes. Nucleotidesequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648to Stalker, and DNA molecules containing these genes are available underATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression ofDNA coding for a glutathione S-transferase is described by Hayes et al.,Biochem. J. 285:173 (1992).

[0169] 3. Genes That Confer or Contribute to a Value-Added Trait, Suchas:

[0170] A. Modified fatty acid metabolism, for example, by transforming aplant with an antisense gene of stearyl-ACP desaturase to increasestearic acid content of the plant. See Knultzon et al., Proc. Natl.Acad. Sci. U.S.A. 89:2624 (1992).

[0171] B. Decreased phytate content

[0172] 1) Introduction of a phytase-encoding gene would enhancebreakdown of phytate, adding more free phosphate to the transformedplant. For example, see Van Hartingsveldt et al., Gene 127:87 (1993),for a disclosure of the nucleotide sequence of an Aspergillus nigerphytase gene.

[0173] 2) A gene could be introduced that reduced phytate content. Inmaize, this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35:383 (1990).

[0174] C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnisα-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Corn Transformation

[0175] Numerous methods for plant transformation have been developed,including biological and physical, plant transformation protocols. See,for example, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation

[0176] One method for introducing an expression vector into plants isbased on the natural transformation system of Agrobacterium. See, forexample, Horsch et al., Science 227:1229 (1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems andmethods for Agrobacterium-mediated gene transfer are provided by Gruberet al., supra, Miki et al., supra, and Moloney et al., Plant CellReports 8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7,1997.

B. Direct Gene Transfer

[0177] Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and corn. Hiei etal., The Plant Journal 6:271-282 (1994) and U.S. Pat. No. 5,591,616issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

[0178] A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299(1988), Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C.,Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

[0179] Another method for physical delivery of DNA to plants issonication of target cells. Zhang et al., Bio/Technology 9:996 (1991).Alternatively, liposome or spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes et al., EMBO J.,4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. U.S.A. 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-omithine have also been reported. Hain etal., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant CellPhysiol. 23:451 (1982). Electroporation of protoplasts and whole cellsand tissues have also been described. Donn et al., In Abstracts of VIIthInternational Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spenceret al., Plant Mol. Biol. 24:51-61 (1994).

[0180] Following transformation of corn target tissues, expression ofthe above-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

[0181] The foregoing methods for transformation would typically be usedfor producing a transgenic inbred line. The transgenic inbred line couldthen be crossed, with another (non-transformed or transformed) inbredline, in order to produce a new transgenic inbred line. Alternatively, agenetic trait which has been engineered into a particular corn lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-eliteinbred line into an elite inbred line, or from an inbred line containinga foreign gene in its genome into an inbred line or lines which do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross, or the process of backcrossing, depending on the context.

[0182] When the term inbred corn plant is used in the context of thepresent invention, this also includes any single gene conversions ofthat inbred. The term single gene converted plant as used herein refersto those corn plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single gene transferred into the inbred via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the gene for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the original inbredof interest (recurrent parent) is crossed to a second inbred(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a cornplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

[0183] The selection of a suitable recurrent parent is an important stepfor a successful backcrossing procedure. The goal of a backcrossprotocol is to alter or substitute a single trait or characteristic inthe original inbred. To accomplish this, a single gene of the recurrentinbred is modified or substituted with the desired gene from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross, one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

[0184] Many single gene traits have been identified that are notregularly selected for in the development of a new inbred but that canbe improved by backcrossing techniques. Single gene traits may or maynot be transgenic, examples of these traits include but are not limitedto, male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Some known exceptions to this are the genes for male sterility,some of which are inherited cytoplasmically, but still act as singlegene traits. Several of these single gene traits are described in U.S.Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, the disclosures of whichare specifically hereby incorporated by reference.

Industrial Applicability

[0185] Corn is used as human food, livestock feed, and as raw materialin industry. The food uses of corn, in addition to human consumption ofcorn kernels, include both products of dry- and wet-milling industries.The principal products of corn dry milling are grits, meal and flour.The corn wet-milling industry can provide corn starch, corn syrups, anddextrose for food use. Corn oil is recovered from corn germ, which is aby-product of both dry- and wet-milling industries.

[0186] Corn, including both grain and non-grain portions of the plant,is also used extensively as livestock feed, primarily for beef cattle,dairy cattle, hogs and poultry.

[0187] Industrial uses of corn include production of ethanol, cornstarch in the wet-milling industry and corn flour in the dry-millingindustry. The industrial applications of corn starch and flour are basedon functional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The corn starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds and other miningapplications.

[0188] Plant parts other than the grain of corn are also used inindustry, for example: stalks and husks are made into paper andwallboard and cobs are used for fuel and to make charcoal.

[0189] The seed of inbred corn line RBO1, the plant produced from theinbred seed, the hybrid corn plant produced from the crossing of theinbred, hybrid seed, and various parts of the hybrid corn plant andtransgenic versions of the foregoing, can be utilized for human food,livestock feed, and as a raw material in industry.

TABLES

[0190] In the tables that follow, the traits and characteristics ofinbred corn line RBO1 are given in hybrid combination. The datacollected on inbred corn line RBO1 is presented for the keycharacteristics and traits. The tables present yield test informationabout RBO1. RBO1 was tested in several hybrid combinations at numerouslocations, with two or three replications per location. Informationabout these hybrids, as compared to several check hybrids, is presented.

[0191] The first pedigree listed in the comparison group is the hybridcontaining RBO1. Information for each pedigree includes:

[0192] 1. Mean yield in Qx/Ha of the hybrid across all locations (MeanYield) is shown in column 2.

[0193] 2. A mean for the percentage moisture (% Moist) for the hybridacross all locations is shown in column 3.

[0194] 3. A mean of the percentage of plants with stalk lodging (%Stalk) across all locations is shown in column 4.

[0195] 4. A mean of the percentage of plants with root lodging (% Root)across all locations is shown in column 5.

[0196] 5. A mean of the stay green across all location from 1 (poor) to9 (good) is shown in column 6.

[0197] 6. Test weight is the grain density measured in kg per 100 litersis shown in column 7.

[0198] 7. A mean of plant height in cm is shown in column 8. TABLE IOverall Comparisons Hybrid vs. Check Hybrids Location: 2000 51 LocationsMini-Strips Mean % % % Stay Test Plant Pedigree Yield Moist Stalk RootGreen Weight Height RBO1* 110.4 17.2 2.1 1.1 70.8 LH185 RBO1* 108.8 18.41.3 0.9 69.5 LH287 At 51 Locations As Com- pared to: Pioneer 107.9 17.34.5 1.8 72.1 Brand 35P12 Pioneer 105.4 17.6 2.2 6.8 73.4 Brand 34G81

[0199] TABLE 2 Overall Comparisons Hybrid vs. Check Hybrids Location:2000 29 Locations UR8E01 Mean % % % Stay Test Plant Pedigree Yield MoistStalk Root Green Weight Height RBO1* 93.8 19.4 3.8 1.4 5 69.4 275 LH185RBO1* 92.8 20.2 6.0 5.9 5 69.8 282 LH287 At 29 Locations As Com- paredto: Pioneer 92.1 20.0 6.9 9.2 6 70.3 280 Brand 35P12 Pioneer 88.8 19.011.0 15.8 4 72.0 293 Brand 34G81

[0200] TABLE 3 Overall Comparisons Hybrid vs. Check Hybrids Location:1999 22 Locations R8E02 Mean % % % Stay Test Plant Pedigree Yield MoistStalk Root Green Weight Height RBO1* 101.6 19 1 0 6 71.0 268 LH185 At 15Locations As Com- pared to: Pioneer 98.1 18.9 2 1 6 74.4 277 Brand 34G81LH198* 94.7 19.7 1 1 7 71.5 252 LH 172

DEPOSIT INFORMATION

[0201] A deposit of the inbred corn seed of this invention is maintainedby AgReliant Genetics, 4640 East State Road 32, Lebanon, Ind. 46052.Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patent andTrademarks to be entitled thereto under 37 CRF 1.14 and 35 USC 122. Uponallowance of any claims in this application, all restrictions on theavailability to the public of the variety will be irrevocably removed byaffording access to a deposit of at least 2.500 seeds of the samevariety with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110.

[0202] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somoclonal variants, variant individuals selected from large populationsof the plants of the instant inbred and the like may be practiced withinthe scope of the invention, as limited only by the scope of the appendedclaims.

What is claimed is:
 1. Seed of corn inbred line designated RBO1,representative seed of said line having been deposited under ATCCAccession No.______.
 2. A corn plant, or parts thereof, produced bygrowing the seed of claim
 1. 3. Pollen of the plant of claim
 2. 4. Anovule of the plant of claim
 2. 5. A corn plant, or parts thereof, havingall of the physiological and morphological characteristics of the cornplant of claim
 2. 6. The corn plant of claim 2, wherein said plant ismale sterile.
 7. A tissue culture of regenerable cells from the cornplant of claim
 2. 8. A tissue culture according to claim 7, the cells orprotoplasts of the tissue culture being from a tissue selected from thegroup consisting of leaves, pollen, embryos, roots, root tips, anthers,silks, flowers, kernels, ears, cobs, husks, and stalks.
 9. A corn plantregenerated from the tissue culture of claim 7, wherein the regeneratedplant is capable of expressing all the morphological and physiologicalcharacteristics of inbred line RBO1.
 10. A corn plant with all of thephysiological and morphological characteristics of corn inbred RBO1,wherein said corn plant is produced by a tissue culture process usingthe corn plant of claim 5 as the starting material for such a process.11. A method for producing a hybrid corn seed comprising crossing afirst inbred parent corn plant with a second inbred parent corn plantand harvesting the resultant hybrid corn seed, wherein said first inbredparent corn plant or second said parent corn plant is the corn plant ofclaim
 2. 12. A hybrid corn seed produced by the method of claim
 11. 13.A hybrid corn plant, or parts thereof, produced by growing said hybridcorn seed of claim
 12. 14. A corn seed produced by growing said cornplant of claim 13 and harvesting the resultant corn seed.
 15. An F₁hybrid seed produced by crossing the inbred corn plant according toclaim 2 with another, different corn plant.
 16. A hybrid corn plant, orits parts, produced by growing said hybrid corn seed of claim
 15. 17. Amethod for producing inbred RBO1, representative seed of which have beendeposited under ATCC Accession No.______, comprising: a) planting acollection of seed comprising seed of a hybrid, one of whose parents isinbred RBO1, said collection also comprising seed of said inbred; b)growing plants from said collection of seed; c) identifying inbredparent plants; d) controlling pollination in a manner which preservesthe homozygosity of said inbred parent plant; and e) harvesting theresultant seed.
 18. The process of claim 17 wherein step (c) comprisesidentifying plants with decreased vigor.
 19. A method for producing aRBO1-derived corn plant, comprising: a) crossing inbred corn line RBO1,representative seed of said line having been deposited under ATCCaccession number ______, with a second corn plant to yield progeny cornseed; and b) growing said progeny corn seed, under plant growthconditions, to yield said RBO1-derived corn plant.
 20. A RBO1-derivedcorn plant, or parts thereof, produced by the method of claim 19, saidRBO1-derived corn plant expressing a combination of at least two RBO1traits selected from the group consisting of: a relative maturity ofapproximately 95 to 113 days, excellent seedling vigor, early pollenshed, excellent brittle stalk resistance, superior root resistance,superior stalk resistance, above average stay green and adapted to theCentral Corn Belt, Northeast, Southeast, Northcentral, Southcentral,Southwest or Western regions of the United States.
 21. The method ofclaim 19, further comprising: c) crossing said RBO1-derived corn plantwith itself or another corn plant to yield additional RBO1-derivedprogeny corn seed; d) growing said progeny corn seed of step (c) underplant growth conditions, to yield additional RBO1-derived corn plants;and e) repeating the crossing and growing steps of (c) and (d) from 0 to7 times to generate further RBO1-derived corn plants.
 22. A furtherRBO1-derived corn plant, or parts thereof, produced by the method ofclaim
 21. 23. The further RBO1-derived corn plant, or parts thereof, ofclaim 22, wherein said further RBO1-derived corn plant, or partsthereof, express a combination of at least two RBO1 traits selected fromthe group consisting of: a relative maturity of approximately 95 to 113days, excellent seedling vigor, early pollen shed, excellent brittlestalk resistance, superior root resistance, superior stalk resistance,above average stay green and adapted to the Central Corn Belt,Northeast, Southeast, Northcentral, Southcentral, Southwest or Westernregions of the United States.
 24. The method of claim 19, still furthercomprising utilizing plant tissue culture methods to derive progeny ofsaid RBO1-derived corn plant.
 25. A RBO1-derived corn plant, or partsthereof, produced by the method of claim 24, said RBO1-derived cornplant expressing a combination of at least two RBO1 traits selected fromthe group consisting of: a relative maturity of approximately 95 to 113days, excellent seedling vigor, early pollen shed, excellent brittlestalk resistance, superior root resistance, superior stalk resistance,above average stay green and adapted to the Central Corn Belt,Northeast, Southeast, Northcentral, Southcentral, Southwest or Westernregions of the United States.
 26. The corn plant, or parts thereof, ofclaim 2, wherein the plant or parts thereof have been transformed sothat its genetic material contains one or more transgenes operablylinked to one or more regulatory elements.
 27. A method for producing acorn plant that contains in its genetic material one or more transgenes,comprising crossing the corn plant of claim 26 with either a secondplant of another corn line, or a non-transformed corn plant of the lineRBO1, so that the genetic material of the progeny that result from thecross contains the transgene(s) operably linked to a regulatory element.28. Corn plants, or parts thereof, produced by the method of claim 27.29. A corn plant, or parts thereof, wherein at least one ancestor ofsaid corn plant is the corn plant of claim 2, said corn plant expressinga combination of at least two RBO1 traits selected from the groupconsisting of: a relative maturity of approximately 95 to 113 days,excellent seedling vigor, early pollen shed, excellent brittle stalkresistance, superior root resistance, superior stalk resistance, aboveaverage stay green and adapted to the Central Corn Belt, Northeast,Southeast, Northcentral, Southcentral, Southwest or Western regions ofthe United States.
 30. A method for developing a corn plant in a cornplant breeding program using plant breeding techniques which includeemploying a corn plant, or its parts, as a source of plant breedingmaterial comprising: using the corn plant, or its parts, of claim 2 as asource of said breeding material.
 31. The corn plant breeding program ofclaim 30 wherein plant breeding techniques are selected from the groupconsisting of: recurrent selection, backcrossing, pedigree breeding,restriction fragment length polymorphism enhanced selection, geneticmarker enhanced selection, and transformation.
 32. A corn plant, orparts thereof, produced by the method of claim
 30. 33. The corn plant ofclaim 5, further comprising a single gene conversion where the geneconfers a characteristic selected from the group consisting of: malesterility, herbicide resistance, insect resistance, resistance tobacterial, fungal or viral disease and corn endosperm or quality.